The identification and analysis of chromosome abnormalities is of great clinical and research importance. Chromosome abnormalities are a significant cause of congenital malformation and are responsible for at least half of spontaneous abortions or miscarriages. Hassold, T., Trends Genet. 2:105 (1986); deGrouchy, J. et al., Clinical Atlas of Human Chromosomes (John Wiley & Sons, New York, 2d ed, 1984); Gardner, R. et al., Chromosome Abnormalities and Genetic Counseling (Oxford Univ. Press, New York, 1989); Gelehrter, T. et al. Principals of Medical Genetics (Williams & Wilkins, Baltimore, Md., 1990) pp. 159-189. The analysis of recurring chromosome abnormalities in malignant cells has also become an integral part of the diagnostic and prognostic workup of many human cancers. Heim, S. et al., Cancer Cytogenetics (A. Liss, New York, 1987); Sandberg, A., The Chromosomes in Human Cancer and Leukemia (Elsevier, New York, 1990); Trent, J. et al., New Eng. J. Med. 322:1508 (1990). In addition to these clinical applications, the molecular examination of chromosome abnormalities has facilitated the identification of genes related to the pathogenesis of both hereditary diseases and cancer. Haluska, F. et al., Ann. Rev. Genet. 21:321 (1987); Weinberg, R., J. NIH Res. 3:45 (1991); Nowell, P. et al., Am. J. Clin. Pathol., 94:229 (1990); Rowley, J., Cancer Res. 50:3816 (1990); Fearon, E. et al., Cell 61:759 (1990); Fountain, J., Science 244:1085 (1989).
Current cytogenetic techniques for chromosome analysis are not, however, entirely satisfactory. One major technical limitation is their inability to unequivocally characterize all cytologically recognizable chromosome rearrangements. For example, amniocentesis may reveal unidentifiable de novo unbalanced translocations or unknown supernumerary marker chromosomes. Warburton, D., J. Hum. Genet. 49:995 (1991); Callen, D. et al., Am J. Hum. Genet. 48:769 (1991). Likewise, in many human cancers, particularly solid tumors, the presence of unidentifiable marker chromosomes or unidentifiable unbalanced translocations frequently prevents complete karyotypic analysis. Mitelman, F., Catalog of Chromosome Aberrations in Cancer (Wiley-Liss, New York,ed. 4th ed, 1991); Mitelman, F. et al., Cytogenet. Cell Genet. 58:1053-1079 (1991).
Chromosome microdissection has become a powerful approach to generate chromosome band-specific libraries and fluorescence in situ hybridization (FISH) probes for physical mapping or cytogenetic analysis. Ludecke, H. J. et al., Nature 338:348-350 (1989); Senger, G. et al., Hum. Genet. 84:507-511 (1990); Kao, F. T., et al., PNAS (USA) 88:1844-1848 (1991); Guan, X. Y. et al., Genomics 14:680-684 (1992); Hirota, T. et al., Genomics 13:349-354 (1992); Meltzer, P. S. et al., Nature Genet. 1:24-28 (1992) and Deng, H. X. et al., Hum. Genet. 89:13-17 (1992). The generation of chromosome region-specific painting probes by polymerase chain reaction (PCR) amplification of microdissected DNA (termed to "Micro-FISH" in Meltzer, P. S. et al., Nature Genet. 1:24-28 (1992) and referred to such herein) has proven useful in solving problems in cytogenetic analysis which are indeterminant by routine chromosome banding analysis, e.g. marker chromosomes. Any abnormal chromosome segment can be microdissected and converted to a FISH probe for hybridization to normal metaphase chromosomes, resulting in a pattern of hybridization which reveals its chromosomal derivation. Meltzer, P. S. et al., Nature Genet. 1:24-28 (1992) and Deng, H. X. et al., Hum. Genet. 89:13-17 (1992). Using this approach, which has been called "reverse chromosome painting," it has been demonstrated that a series of chromosomes with apparent terminal deletions were actually cryptic unbalanced translocations. Meltzer, P. S. et al., Nature Genet. (in press) (1993).
In principle, Micro-FISH has made it possible to identify the chromosomal constitution of any cytologically visible chromosome rearrangement. However, previously published microdissection techniques have included the time-consuming and labor-intensive requirement of dissecting 20-40 DNA fragments from a target region in order to obtain sufficient template for PCR amplification. Guan, X. Y. et al., Genomics 14:680-684 (1992)and Meltzer, P. S. et al., Nature Genet. 1:24-28 (1992). Several hours are required for the microdissection procedure, and with the addition of each copy to the collection drop, the probability of extraneous DNA contamination is increased. The contaminating DNA may derive from the glass microneedle which touches DNA other than target DNA, or could be introduced into the collection drop from the air when the tube is repeatedly opened and closed. DNA contamination is a critical problem in the amplification of dissected DNA because the initial amount of dissected material is exceedingly small (in the range of 10.sup.-13 to 10.sup.-14 g/fragment). Accordingly, even minute amounts of contaminating DNA can overwhelm the microdissected DNA leading to a useless amplification product. Unfortunately, decreasing the copy number of dissected DNA fragments tends to reduce the complexity and, therefore, the signal intensity of the resulting FISH probe.
It would thus be desirable to provide a method of generating region-specific probes for any chromosome band or region. It would further be desirable that the method for the preparation of such probes be relatively simple, rapid and reproducible. It would also be desirable to provide a method wherein a large number of highly specific probes could be easily generated. It would further be desirable to provide a method of generating region-specific probes which decreases the number of microdissected chromosme fragments required for probe generation. It would also be desirable to provide a method of generating region-specific probes which requires less time and labor than is required in current methods. It would further be desirable to provide a method of generating region-specific probes which reduces the risk of exogenous DNA contamination. It would also be desirable that probes generated by this method provide means for identifying all cytologically recognizable chromosomal rearrangements and deletions. It would further be desirable to provide a method of screening genomic, recombinant or other DNA libraries with a region-specific probe.